| Project/Area Number |
10660085
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Osaka University |
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Principal Investigator |
MATSUURA Yoshiki Inst. for Protein Res., Osaka Univ., Assoc.Prof., 蛋白質研究所, 助教授 (90029968)
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| Co-Investigator(Kenkyū-buntansha) |
KATSUGA Yoshio Hyogo Industry Res. Center, Principal Researcher, 主任研究員
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Isoamylase / Structure / Substrate complexed / X-ray Analysis / 基質結合 / X線結晶解析 |
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| Research Abstract |
The three dimensional structure of isoamylase was determined at 2.2 Å resolution.
In order to understand the substrate specificity and catalytic mechanism of isoamylase, we have carried out the X-ray crystal structure analysis of enzyme-inhibitor complex of Pseudomonas isoamylase. Complexed crystals with maltotetraose that is a competitive inhibitor were prepared by soaking technique.
Diffraction data were collected for the complexed crystals with an imaging plate diffractometer Rigaku R-AXIS4 at the experimental hutch A of Hyoso beamline (BL24XU). For the complexed crystals, 233, 121 reflections were observed and 79, 074 independent reflections were obtained with a merging R-value of 0.092. The diffraction data set was 74.4% complete at the resolution of 1.8 Å.
The molecular replacement method was applied for each diffraction data by using the program X-PLOR.The positions of the inhibitor were investigated in the difference Fourier maps. Finally, we could identify four positions of glucose residues near the active residues. These positions of glucose residues correspond to the -2 to -4 subsites in α-amylases. The isoamylase molecule with 526 water oxygens and maltotetraose was refined at 1.8 Å resolution by using the program X-PLOR.The crystallographic R-factor and Rfree-factor for the model was converged to 0.201 and 0.263, respectively. From structural comparison between the isoamylase-maltotetraose complex and α-amylase-inhibitor complexes, it is clear that streochemical repulsion between Leu376 of isoamylase and Glc(+1) residue occur in a complex between isoamylase and linear substrate composed with α-1,4-glucosidic linkages.
Thus, we conclude that Leu376 is the essential residue for substrate specificity in isoamylase activity.
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